adequate recovery of nucleated cells and progenitors to enable engraftment. .... 50,000) (USB Corporation Cleveland, OH,USA). Final volumes of UCB units with ...
Biomedical Research 2012; 23 (3): 395-401
ISSN 0970-938X Scientific Publishers of India
Evaluation of procedures for quantification of CD34+haematopoietic stem cells and viability studies in umbilical cord blood derived mononuclear cells. Sheeba Afreen1,2, Tulika Chandra1, Ashutosh Kumar2, Uma Singh3, Ashish Gupta1,2 1
Department of Transfusion Medicine and Blood Bank, 2Department of Pathology, 3Department of Obstetrics and Gynecology, Chhattrapati Shahuji Maharaj Medical University ( Earlier King Georg’s Medical University), Lucknow Uttar Pradesh, India
Abstract Umbilical cord blood (UCB) transplantation is being used as an alternative source of haematopoietic stem cells for bone marrow reconstitution. Umbilical cord blood (UCB) transplantation is being used as an alternative source of haematopoietic stem cells for bone marrow reconstitution. Separation and processing of UCB samples in large numbers for storage in cord blood banks ideally needs to be partially automated. This study examines the loss of CD34+cells concentration and viability of mononuclear cells from baseline verses one month, baseline verses six month and one month verses six month as well as standardization of procedure for cryopresevation of cord blood derived haematopoietic stem cells. A total of 500 umbilical cord blood units were collected. Samples were analyzed for CD34+ cells concentration and viability of mononuclear cells at baseline, one month and six month. In present study we found that the mean CD34+cell loss from base line to one month was found to be 0.24℅, baseline to six month was 0.78℅ and one month to six month was 0.54℅. The mean viability of mononuclear cell loss from base line to one month was found to be 8.26℅, baseline to six month was 74.20℅ and one month to six month was8.99℅. Our data indicate that CD34+ cells and viability of mononuclear cells in UCB were maintained after cryopreservation of six month period in -800C. This amount of CD34+ cells were within the range used for successful engraftment in both related and unrelated cord blood transplantation. Keywords: Umbilical cord blood, CD34+ cells, mononuclear cells, Viability, Cryopreservation Accepted February 22 2012 Introduction Umbilical cord blood contains hematopoieticstem/ progenitor cells that have proven useful clinically to reconstitute the hematopoietic system in children and some adults [1]. Work that was begun in the early 1980s revealed that cord blood (ie, the leftover blood in the umbilical cord and placenta after the birth of a child) was comparable to bone marrow in terms of its utility in stem cell transplantation [2]. Cord blood offers a number of advantages over the bone marrow including a lower incidence of Graft verses host disease and less strict HLA-matching requirements, which could increase its availability to transplant patients [3]. During the past 10 years, clinical use of cord blood (with more than 8,000 transplants worldwide) has shown that it is a suitable alternative to bone marrow [4]. In addBiomedical Research 2012 Volume 23 Issue 3
ition to its use as a substitute for bone marrow, cord blood has recently been used in a variety of regenerative medicine applications. Work done by Harris and colleagues (2007) has shown that cord blood contains a mixture of pluripotent stem cells capable of giving rise to cells derived from the endodermal, mesodermal, and ectodermal lineages [5]. Thus, cord blood appears to be a practical substitute for embryonic stem cells and readily available for use in tissue engineering and regenerative medicine. The cryopreservation process is of importance for all types of stem cell collection, but it is perhaps particularly critical for umbilical cord blood (UCB). The actual transplant is harvested at the time of birth and used at a later point in time for often an indeterminate recipient [6]. Cryopreservation of cord blood derived haematopoietic stem cells is critical for UCB banking, transplantation as 395
Afreen/Chandra/Kumar/Singh/Gupta well as for research applications by providing readily available specimens. Separation and processing of UCB samples in large number of storage in cord blood banks ideally needs to be partially automated to allow large numbers of samples to be processed efficiently. A closed system reduces the risk of bacterial contamination after collection. The processing method must allow for the adequate recovery of nucleated cells and progenitors to enable engraftment. Early attempts at separating cord blood by density gradient techniques led to loss of mononuclear cells which suggests that cord blood should be stored unseparated. However, volume reduction is essential for cord blood banks to be economical and efficient. Several different methods have been employed to reduce the volume prior to cryopreservation without loss of progenitor cells such as density gradient separation [7], sedimentation of red cells by gelatin [8], rouleaux formation induced by hydroxyethyl starch (HES) and centrifugation [9], and differential centrifugation with expression of RBC and plasma [10]. The present study was carried out to assess loss of CD34+cells concentration and viability of mononuclear cells from baseline verses one month, baseline verses six month and one month verses six month as well as standardization of procedure for cryopresevation of cord blood derived haematopoietic stem cells.
Materials and Methods A total of 480 were obtained from both vaginal and caesarian deliveries from the Department of Obstetrics and Gynecology. Processing of 480 samples of UCB was done in the Department of Transfusion Medicine, Chattrapati Shahuji Maharaj Medical University, Lucknow. Written informed consent was obtained from mother. The study has been approved by Institutional Ethical Committee of Chattrapati Shahu Ji Maharaj Medical University, Lucknow. Inclusion Criteria All healthy full term females with no history hepatitis, infectious disease, diabetes mellitus, severe hypertension, abortion or bad obstetrics. Infants delivered at term (3141 weeks) were included.
A) Collection of UCB Cord blood was collected from 370 (77.08%) normal vaginal and 110 (22.9%) cesarean deliveries after the completion of delivery before placenta expulsion in CPDA triple blood bag containing 69 ml anticoagulant citrate, phosphate, dextrose and adenine. 20 ml anticoagulant was removed before collection of umbilical cord blood. After the delivery of the baby the cord was clamped and wiped with alcohol or betadine to ensure sterility of the collection. A needle was inserted into the umbilical vein above the clamp. The blood was drained via gravity into the sterile collection bag, containing Citrate Phosphate Dextrose Adenine (CPDA) as an anticoagulant. Efforts were made to obtain maximal volumes from each collection. The umbilical cord blood units were stored at 40C and processed within 24 hours. Samples of 3 ml per unit were taken at this stage for nucleated cell (NC) count. B) Processing of UCB Processing was done within 24 hours of cord blood collection. The CD34+ cell concentration, total nucleated cells count and viability assay were done on all the samples. Transmissible disease testing for Human Immunodeficiency Virus (HIV-1and 2), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) and syphilis was also performed. In the processing of samples, which included determination of cord blood volumes. Determination of initial level of total nucleated cells (TNC) and mononuclear cells (MNC) before centrifugation was done. The UCB product was mixed with HES containing solution (6% HES in 0.9 NaCl) in a 5: 1 ratio and centrifuged in a Cryofuge 6000i (Heraeus-Kendro, Hanau, Germany) at 1200 x g for 10 min. The WBC-rich plasma was expressed in a separate bag and again centrifuged at 2500 x g for 10 min. The WBC poor plasma was expressed and discarded. The remaining suspension of mononuclear cells was left whose counts were recorded. The complete process was performed in a closed system with the use of a sterile connecting device (Terumo TSCD, SC-201 AH, Leuven, Belgium).
All females with APH, eclampsia and high risk cases were excluded from the study.
C) Assessment of Total nucleated count, CD34+ and viability test The samples were analyzed for CD34+cells concentration and viability of mononuclear cell on baseline period, one month and six month. The total nucleated cells count was done before RBC depletion by automated cell counter (Sysmex KX-21, Japan). CD34+ cells concentration was done by flowcytometery and viability of mononuclear cells were done by trypan blue dye exclusion test.
Cord blood donor infants Birth weight, baby sex, mode of delivery and gestational age of the baby were also recorded. After collection, the cord blood was sent in the transport boxes to the department of Transfusion Medicine, Chattrapati Shahuji Maharaj Medical University, Lucknow, India.
D) Cryopreservation Dimethyl sulfoxide (DMSO) (Merck Limited, Mumbai) was used at a final concentration of 10% (vol/vol). The required volume of sterile, chilled DMSO solution was added to the blood bag over the course of 15 min by using a syringe pump and an orbital mixer to assure smooth but
Exclusion Criteria
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CD34+ cells concentration in umbilical cord blood vigorous mixing. In these experiments, UCB processed units were mixed with either 20% DMSO in saline or 50% DMSO in 5% (wt/vol) Dextran 40 (Mr 35,00050,000) (USB Corporation Cleveland, OH,USA). Final volumes of UCB units with DMSO was a uniform 25 ml. Cryoprotectant UCB units were kept cold with wet ice throughout the addition. When the concentration of DMSO reached 10%, cell suspensions were transferred to -200C for 5-10 hours. Subsequently it was transferred to 400C for 4 hours and then stored at -80 0C for six month. E) Thawing and Washing Umbilical cord blood processing method was done as previously described by Rubinstein and colleagues (New York Blood Center) [13]. UCB units were thawed and washed. Briefly, umbilical cord blood units were taken out of the -800C and immersed in a 370C water bath. After inspection, 10 % dextran 40 was slowly added followed by 5 percent human albumin and the bag was left to equilibrate for 5 minutes. The product was transferred to a first transfer bag and centrifuged at 400 x g for 15 minutes at 40C. Cell pellet were obtained in first transfer bag and approximately three-fourths of the wash supernatant was expressed and transferred in a second transfer bag. The wash supernatant in the second transfer was again centrifuged at 800 x g for 15 minutes at 40C. Three fourth of the wash supernatant was expressed. The residual cell pellets obtained in the second transfer bag were combined in first transfer bag through sterile tube welder. The combined cell pellets were resuspended in 10 percent dextran and 5 percent human albumin. Statistical Analysis Data collected was entered in Microsoft excel and checked for any inconsistency. The mean and standard deviation were calculated at baseline, one month and six months for CD34+ cells concentration and viability of mononuclear cells count. The repeated measures of analysis (ANOVA) was used to compare loss from baseline, one month and six months and paired t-test was used for pairwise comparisons. The mean difference with its 95% confidence interval was calculated. The percentage loss was also calculated. The p-value < 0.05 was considered significant. All the analysis was carried out using spss 15.0 version.
Results A total of 480 samples of UCB were analyzed. Considering newborn sex distribution, out of 268 infants (55.8%) were males and 212 (44.6%) were females. The median gestational age was 38 wks (mean 37.57 ±1.82 wks, range 31 – 41).Out of 480 cord blood donors 150 (31.2%) females were of small gestational age (SGA) and 330 (68.7%) were of large gestational age (LGA). The distribution of birth weight was normal (mean 2790 ± 426 Biomedical Research 2012 Volume 23 Issue 3
gram, range 1800-3900). 120 (25.0%) baby have weight below 2500 and 360 (75.0%) baby have 2500 to 3900 gram (Table-1). CD34+ cell concentration (%) In the baseline, CD34+ cells concentration was 1.94 ℅ (±0.90) which decreased at one month to 1.70 ℅ (±0.89) which decreased at six months to 1.16℅ (±0.87) (Table2). After thawing and washing the mean CD34+ cell loss from base line to one month was found to be 0.24℅ (95% CI: 0.23-0.25), which was statistically significant (p
